P P Ins(1,4,5)P3 Ca++ SR Ca++ CaM MLCK MLC MLC P GEFs Rho Rho-kinase CPI-17 CPI-17 MBS M20 cat MBS Inactive Active Contraction M20 cat MP Y27632 GTP ? AA PLC GqG12/13 •Fig. 14.8 Rho-kinase(ROK)signalinginsmoothmuscle.A,AvarietyofagonistsofG-coupledreceptorssimultaneouslystimulateInsP3productionandactivateRhoA-ROKsignaling.InsP3(Ins[1,4,5]P3)isproducedbyphospholipaseC(PLC)-mediatedhydrolysisofPIP2.InsP3increasesintracellular[Ca++]byopeningInsP3-gatedCa++ channelsinthesarcoplasmicreticulum(SR),therebyresultinginCa++-calmodulin–dependentactivationofmyosinlight-chainkinase(MLCK)andsubsequentphosphorylationofthemyosinregulatorylightchain(MLC)andpromotionofactin-myosininteraction(contraction).ActivatedRhoA(depictedasRho-GTP)stimulatesROK,whichinhibitsmyosinphosphatase(MP)byphosphorylatingthemyosin-bindingsubunit(MBS)ofMP.ROKalsoinhibitsMPindirectlybyphosphorylating/activatingCPI-17,a17-kDainhibitorofMP.TheneteffectofROKphosphorylationsisadecreaseinMPactivity,whichresultsinanincreasedlevelofmyosinlight-chainphosphorylation(MLCP)andhencegreaterforceofcontractionatagivenintracellular[Ca++](i.e.,increasedsensitivityofcontractiontoCa++).B,Ca++ sensitization referstoanincreaseintheforceofcontractionatagivenintracellular[Ca++]andisdepictedasaleftwardshiftintheCa++ dependenceofcontractionofsmoothmuscle.Ca++ sensitizationcanresultfromadecreaseinMPactivityand/oranincreaseinMLCKactivityatagivenintracellular[Ca++](asshownintheROKsignalingcascadeinpanelA).Conversely,Ca++ desensitization referstoadecreaseintheforceofcontractionatagivenintracellular[Ca++]andisdepictedasarightwardshiftintheCa++ dependenceofcontraction.AA,arachidonicacid;CaM,Ca++-calmodulincomplex;cat,catalyticsubunitofmyosinphosphatase;G12/13andGq,heterotrimericG-proteins;GEF,guanine-nucleotideexchangefactor;M20,subunitofmyosinphosphatase;MP,myosinphosphatasecomplex;Y27632,commercialinhibitorofROK.(FromFukataYetal.Trends Pharmacol Sci 2001;22:32-39.) •Fig. 14.9 Timecourseofeventsincross-bridgeactivationandcontractioninsmoothmuscle.A,AbriefperiodofstimulationisassociatedwithCa++ mobilization,followedbycross-bridgephosphorylationandcyclingtoproduceabriefphasic,twitch-likecontraction.B,Inasustainedtoniccontractionproducedbyprolongedstimulation,theCa++ andphosphorylationlevelstypicallyfallfromaninitialpeak.Forceismaintainedduringtoniccontractionsatareduced[Ca++](andhencealowlevelofmyosinlight-chainphosphorylation),withlowercross-bridgecyclingratesmanifestedbylowershorteningvelocitiesandATPconsumption.